* We then ligated the samples with concentrations of 4:4, 0.5:7.5, and 7.5:0.5 micro liters of plasmid DNA to our insert DNA (OmcF DNA). We also did 2 controls for each 450 and 350 DNA sample. One with no plasmid DNA and one with no insert DNA

+

* We then ligated the samples with concentrations of 4:4, 0.5:7.5, and 7.5:0.5μL of plasmid DNA to our insert DNA (OmcF DNA). We also did 2 controls for each 450 and 350 DNA sample. One with no plasmid DNA and one with no insert DNA

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* On Thursday, we continued to work on the transformation, by actually combining our ligated samples with 40 micro liters of E.coli cells, icing, heat shocking, then icing the samples. We then added SOC and shook the samples for an hour.

+

* On Thursday, we continued to work on the transformation, by actually combining our ligated samples with 40μL of E.coli cells, icing, heat shocking, then icing the samples. We then added SOC and shook the samples for an hour at 37°C.

* We then plated our samples on solid media plates.

* We then plated our samples on solid media plates.

Revision as of 17:38, 15 November 2012

Cloning E.coli with the OmcF gene

On Tuesday, we worked on the digestion of our plasmid DNA with the restriction enzymes SpecI and PstI and 10x Digestion Buffer. * We then purified the sample with the PCR purification kit to rid the sample of any additional parts.

To check the approximate quantity of our samples, we ran a gel along with a Low Range Ladder to judge the amounts of each sample. After this we determine the appropriate concentrations to use during ligation to help aid that process.

They were loaded into the well as follows:

100bp ladder

2x plasmid DNA

2x plasmid DNA

4μL Low Range Ladder

2μL Low Range Ladder

450bp 2x DNA

450bp 1x DNA

350bp 2x DNA

350bp 1x DNA

100bp Ladder

We then ligated the samples with concentrations of 4:4, 0.5:7.5, and 7.5:0.5μL of plasmid DNA to our insert DNA (OmcF DNA). We also did 2 controls for each 450 and 350 DNA sample. One with no plasmid DNA and one with no insert DNA

On Thursday, we continued to work on the transformation, by actually combining our ligated samples with 40μL of E.coli cells, icing, heat shocking, then icing the samples. We then added SOC and shook the samples for an hour at 37°C.